Not all J domains are created equal: implications for the specificity of Hsp40-Hsp70 interactions
- Hennessy, Fritha, Nicoll, Willam S, Zimmerman, Richard, Cheetham, Michael E, Blatch, Gregory L
- Authors: Hennessy, Fritha , Nicoll, Willam S , Zimmerman, Richard , Cheetham, Michael E , Blatch, Gregory L
- Date: 2005
- Language: English
- Type: Article
- Identifier: vital:6487 , http://hdl.handle.net/10962/d1006270 , http://dx.doi.org/10.1110/ps.051406805
- Description: Heat shock protein 40s (Hsp40s) and heat shock protein 70s (Hsp70s) form chaperone partnerships that are key components of cellular chaperone networks involved in facilitating the correct folding of a broad range of client proteins. While the Hsp40 family of proteins is highly diverse with multiple forms occurring in any particular cell or compartment, all its members are characterized by a J domain that directs their interaction with a partner Hsp70. Specific Hsp40-Hsp70 chaperone partnerships have been identified that are dedicated to the correct folding of distinct subsets of client proteins. The elucidation of the mechanism by which these specific Hsp40-Hsp70 partnerships are formed will greatly enhance our understanding of the way in which chaperone pathways are integrated into finely regulated protein folding networks. From in silico analyses, domain swapping and rational protein engineering experiments, evidence has accumulated that indicates that J domains contain key specificity determinants. This review will critically discuss the current understanding of the structural features of J domains that determine the specificity of interaction between Hsp40 proteins and their partner Hsp70s. We also propose a model in which the J domain is able to integrate specificity and chaperone activity.
- Full Text:
- Date Issued: 2005
- Authors: Hennessy, Fritha , Nicoll, Willam S , Zimmerman, Richard , Cheetham, Michael E , Blatch, Gregory L
- Date: 2005
- Language: English
- Type: Article
- Identifier: vital:6487 , http://hdl.handle.net/10962/d1006270 , http://dx.doi.org/10.1110/ps.051406805
- Description: Heat shock protein 40s (Hsp40s) and heat shock protein 70s (Hsp70s) form chaperone partnerships that are key components of cellular chaperone networks involved in facilitating the correct folding of a broad range of client proteins. While the Hsp40 family of proteins is highly diverse with multiple forms occurring in any particular cell or compartment, all its members are characterized by a J domain that directs their interaction with a partner Hsp70. Specific Hsp40-Hsp70 chaperone partnerships have been identified that are dedicated to the correct folding of distinct subsets of client proteins. The elucidation of the mechanism by which these specific Hsp40-Hsp70 partnerships are formed will greatly enhance our understanding of the way in which chaperone pathways are integrated into finely regulated protein folding networks. From in silico analyses, domain swapping and rational protein engineering experiments, evidence has accumulated that indicates that J domains contain key specificity determinants. This review will critically discuss the current understanding of the structural features of J domains that determine the specificity of interaction between Hsp40 proteins and their partner Hsp70s. We also propose a model in which the J domain is able to integrate specificity and chaperone activity.
- Full Text:
- Date Issued: 2005
Characterisation of the J domain aminoacid residues important for the interaction of DNAJ-like proteins with HSP70 chaperones
- Authors: Hennessy, Fritha
- Date: 2004
- Subjects: Heat shock proteins Protein folding Proteins -- Analysis Proteins -- Structure Molecular chaperones
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3937 , http://hdl.handle.net/10962/d1003996
- Description: The 70 kDa heat shock proteins (Hsp70s) are vital for normal protein folding, as they stabilise the unfolded state of nascent polypeptides, allowing these sufficient time to attain a correct tertiary structure. Hsp70s are aided by the DnaJ-like family of proteins, which interact with Hsp70s in order to enhance the chaperone activity of these proteins. DnaJ-like proteins contain a J domain, a seventy amino acid domain consisting of four α-helices, which is defined by the presence of an invariant tripeptide of histidine, proline and aspartic acid (HPD motif). This motif is key to the interaction between DnaJ-like proteins and Hsp70s. This thesis has focused on determining the presence of other conserved residues in the J domain and their role in mediating the interaction of DnaJ-like proteins with partner Hsp70s. DnaJ-like proteins from Agrobacterium tumefaciens RUOR were isolated and used as a model system. A. tumefaciens DnaJ (Agt DnaJ) was able to replace the lack of E. coli DnaJ in an E. coli null mutant strain, however, additional A. tumefaciens DnaJ-like proteins Agt DjC1/DjlA, Agt DjC2 and Agt DjC5 were unable to complement for the lack of E. coli DnaJ. Replacement of the Agt DnaJ J domain with J domains from these proteins resulted in non-functional chimeric proteins, despite some sequence conservation. The kinetics of the basal specific ATPase activity of Agt DnaK, and its ability to have this activity stimulated by Agt DnaJ and Agt DnaJ-H33Q were also investigated. Stimulation of the ATPase activity by Agt DnaJ ranged between 1.5 to 2 fold, but Agt DnaJ-H33Q was unable to stimulate the basal ATPase activity. Conserved amino acids in the J domain were identified in silico, and these residues were substituted in the J domain of Agt DnaJ. The ability of these derivative proteins to replace E. coli DnaJ was investigated. Alterations in the HPD motif gave rise to proteins unable to complement for lack of E. coli DnaJ, consistent with literature. Agt DnaJ-R26A was unable to replace E. coli DnaJ suggesting that Arg26 could be key to the interaction with partner Hsp70s. Agt DnaJ-D59A was unable to replace E. coli DnaJ; substitutions in Asp59 have not previously been shown to impact on the function of DnaJ. Substituting Arg63 in Agt DnaJ abrogated the levels of complementation. Substitution of several structural residues was also found to disrupt the in vivo function of Agt DnaJ suggesting that the maintenance of the structural integrity of the J domain was important for function. This study has identified a number of residues critical to the structure and function of the J domain of Agt DnaJ, and potentially of general importance as molecular determinants for DnaJ-Hsp70 interaction.
- Full Text:
- Date Issued: 2004
- Authors: Hennessy, Fritha
- Date: 2004
- Subjects: Heat shock proteins Protein folding Proteins -- Analysis Proteins -- Structure Molecular chaperones
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3937 , http://hdl.handle.net/10962/d1003996
- Description: The 70 kDa heat shock proteins (Hsp70s) are vital for normal protein folding, as they stabilise the unfolded state of nascent polypeptides, allowing these sufficient time to attain a correct tertiary structure. Hsp70s are aided by the DnaJ-like family of proteins, which interact with Hsp70s in order to enhance the chaperone activity of these proteins. DnaJ-like proteins contain a J domain, a seventy amino acid domain consisting of four α-helices, which is defined by the presence of an invariant tripeptide of histidine, proline and aspartic acid (HPD motif). This motif is key to the interaction between DnaJ-like proteins and Hsp70s. This thesis has focused on determining the presence of other conserved residues in the J domain and their role in mediating the interaction of DnaJ-like proteins with partner Hsp70s. DnaJ-like proteins from Agrobacterium tumefaciens RUOR were isolated and used as a model system. A. tumefaciens DnaJ (Agt DnaJ) was able to replace the lack of E. coli DnaJ in an E. coli null mutant strain, however, additional A. tumefaciens DnaJ-like proteins Agt DjC1/DjlA, Agt DjC2 and Agt DjC5 were unable to complement for the lack of E. coli DnaJ. Replacement of the Agt DnaJ J domain with J domains from these proteins resulted in non-functional chimeric proteins, despite some sequence conservation. The kinetics of the basal specific ATPase activity of Agt DnaK, and its ability to have this activity stimulated by Agt DnaJ and Agt DnaJ-H33Q were also investigated. Stimulation of the ATPase activity by Agt DnaJ ranged between 1.5 to 2 fold, but Agt DnaJ-H33Q was unable to stimulate the basal ATPase activity. Conserved amino acids in the J domain were identified in silico, and these residues were substituted in the J domain of Agt DnaJ. The ability of these derivative proteins to replace E. coli DnaJ was investigated. Alterations in the HPD motif gave rise to proteins unable to complement for lack of E. coli DnaJ, consistent with literature. Agt DnaJ-R26A was unable to replace E. coli DnaJ suggesting that Arg26 could be key to the interaction with partner Hsp70s. Agt DnaJ-D59A was unable to replace E. coli DnaJ; substitutions in Asp59 have not previously been shown to impact on the function of DnaJ. Substituting Arg63 in Agt DnaJ abrogated the levels of complementation. Substitution of several structural residues was also found to disrupt the in vivo function of Agt DnaJ suggesting that the maintenance of the structural integrity of the J domain was important for function. This study has identified a number of residues critical to the structure and function of the J domain of Agt DnaJ, and potentially of general importance as molecular determinants for DnaJ-Hsp70 interaction.
- Full Text:
- Date Issued: 2004
Molecular chaperones in biology, medicine and protein biotechnology
- Boshoff, Aileen, Nicoll, William S, Hennessy, Fritha, Ludewig, M H, Daniel, Sheril, Modisakeng, Keoagile W, Shonhai, Addmore, McNamara, Caryn, Bradley, Graeme, Blatch, Gregory L
- Authors: Boshoff, Aileen , Nicoll, William S , Hennessy, Fritha , Ludewig, M H , Daniel, Sheril , Modisakeng, Keoagile W , Shonhai, Addmore , McNamara, Caryn , Bradley, Graeme , Blatch, Gregory L
- Date: 2004
- Language: English
- Type: Article
- Identifier: vital:6457 , http://hdl.handle.net/10962/d1004479
- Description: Molecular chaperones consist of several highly conserved families of proteins, many of which consist of heat shock proteins. The primary function of molecular chaperones is to facilitate the folding or refolding of proteins, and therefore they play an important role in diverse cellular processes including protein synthesis, protein translocation, and the refolding or degradation of proteins after cell stress. Cells are often exposed to different stressors, resulting in protein misfolding and aggregation. It is now well established that the levels of certain molecular chaperones are elevated during stress to provide protection to the cell. The focus of this review is on the impact of molecular chaperones in biology, medicine and protein biotechnology, and thus covers both fundamental and applied aspects of chaperone biology. Attention is paid to the functions and applications of molecular chaperones from bacterial and eukaryotic cells, focusing on the heat shock proteins 90 (Hsp90), 70 (Hsp70) and 40 (Hsp40) classes of chaperones, respectively. The role of these classes of chaperones in human diseases is discussed, as well as the parts played by chaperones produced by the causative agents of malaria and trypanosomiasis. Recent advances have seen the application of chaperones in improving the yields of a particular target protein in recombinant protein production. The prospects for the targeted use of molecular chaperones for the over-production of recombinant proteins is critically reviewed, and current research on these chaperones at Rhodes University is also discussed.
- Full Text:
- Date Issued: 2004
- Authors: Boshoff, Aileen , Nicoll, William S , Hennessy, Fritha , Ludewig, M H , Daniel, Sheril , Modisakeng, Keoagile W , Shonhai, Addmore , McNamara, Caryn , Bradley, Graeme , Blatch, Gregory L
- Date: 2004
- Language: English
- Type: Article
- Identifier: vital:6457 , http://hdl.handle.net/10962/d1004479
- Description: Molecular chaperones consist of several highly conserved families of proteins, many of which consist of heat shock proteins. The primary function of molecular chaperones is to facilitate the folding or refolding of proteins, and therefore they play an important role in diverse cellular processes including protein synthesis, protein translocation, and the refolding or degradation of proteins after cell stress. Cells are often exposed to different stressors, resulting in protein misfolding and aggregation. It is now well established that the levels of certain molecular chaperones are elevated during stress to provide protection to the cell. The focus of this review is on the impact of molecular chaperones in biology, medicine and protein biotechnology, and thus covers both fundamental and applied aspects of chaperone biology. Attention is paid to the functions and applications of molecular chaperones from bacterial and eukaryotic cells, focusing on the heat shock proteins 90 (Hsp90), 70 (Hsp70) and 40 (Hsp40) classes of chaperones, respectively. The role of these classes of chaperones in human diseases is discussed, as well as the parts played by chaperones produced by the causative agents of malaria and trypanosomiasis. Recent advances have seen the application of chaperones in improving the yields of a particular target protein in recombinant protein production. The prospects for the targeted use of molecular chaperones for the over-production of recombinant proteins is critically reviewed, and current research on these chaperones at Rhodes University is also discussed.
- Full Text:
- Date Issued: 2004
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